Intranasal antisepsis to reduce influenza virus transmission in an animal model

Abstract Background Seasonal influenza annually causes significant morbidity and mortality, and unpredictable respiratory virus zoonoses, such as the current COVID‐19 pandemic, can threaten the health and lives of millions more. Molecular iodine (I2) is a broad‐spectrum, pathogen‐nonspecific antiseptic agent that has demonstrated antimicrobial activity against a wide range of bacteria, virus, and fungi. Methods We investigated a commercially available antiseptic, a non‐irritating formulation of iodine (5% povidone‐iodine) with a film‐forming agent that extends the duration of the iodine's antimicrobial activity, for its ability to prevent influenza virus transmission between infected and susceptible animals in the guinea pig model of influenza virus transmission. Results We observed that a once‐daily topical application of this long‐lasting antiseptic to the nares of either the infected virus‐donor guinea pig or the susceptible virus‐recipient guinea pig, or to the nares of both animals, prior to virus inoculation effectively reduced transmission of a highly transmissible influenza A virus, even when the donor and recipient guinea pigs shared the same cage. Daily treatment of the recipient guinea pig starting 1 day after initial exposure to an infected donor guinea pig in the same cage was similarly effective in preventing detectable influenza virus infection in the recipient animal. Conclusions We conclude that a daily application of this antiseptic formulation is efficacious in reducing the transmission of influenza A virus in the guinea pig model, and further study in this and other preclinical models is warranted.

respiratory disease characterized by fever, chills, lethargy, headache, cough, sore throat, and runny nose to severe pneumonia that may be fatal, particularly in elderly persons and in those with immunosuppressive conditions. 4,5 The main strategy for prevention and control of seasonal influenza has been vaccination. However, influenza vaccines are not optimally protective against influenza virus infection, 6 and vaccine uptake is incomplete. In the United States, vaccination rates have not significantly changed since 2013, ranging from 41.7% in [2015][2016] to an estimated 37.1% in 2017-2018 among adults. 7 Additionally, current influenza vaccine technologies cannot be preemptively deployed in anticipation of pandemic influenza; instead, they can be implemented only in response to a pandemic that is already underway.
Nonpharmaceutical interventions (NPIs), such as mask wearing, hand hygiene, indoor ventilation, quarantine of symptomatic individuals, and social distancing for asymptomatic persons are variably effective at preventing the spread of influenza and other respiratory viruses, [8][9][10] including pandemic SARS-CoV-2, [11][12][13][14] although the simultaneous implementation of multiple NPIs may be more effective than any one NPI alone. 9,12 Additional pathogen-nonspecific interventions that are effective at hindering or preventing person-to-person disease transmission and that can be implemented in concert with other NPIs may help to mitigate the impact of future zoonoses like COVID-19 or pandemic influenza.
The use of iodine as a topical antiseptic dates back nearly as far as its discovery in 1811 15 and predates the widespread understanding of germ theory. 16 Iodine-based antiseptics are broadly antimicrobial; they are active against bacteria, fungi, mycobacteria, protozoa, and viruses, including influenza virus. 17,18 The inhalation of iodine vapors to treat various respiratory diseases was proposed as early as 1829, 19 and topical, inhalational, and oral iodine preparations were used empirically to prevent influenza during the 1918 "Spanish flu" [20][21][22][23] and the 1957 "Asian flu" 24 pandemics. In experiments conducted in the 1940s, the application of an ethanol-based tincture of iodine to the snouts of mice prevented disease upon exposure to a dose of aerosolized influenza virus lethal to control mice. 25 Subsequently, povidone-iodine (PVP-I) was introduced in the 1950s as a novel iodine-based antiseptic. PVP-I is a complex of the polymer povidone (polyvinylpyrrolidone) and triiodide (I 3 À ) that does not require additional iodine solubilizers and is less irritating to the skin than tincture of iodine while retaining its broad-spectrum antimicrobial activity. 17,18 Because aqueous solutions of PVP-I maintain a low but constant concentration of free iodine (the active antimicrobial agent) in dynamic equilibrium with the PVP-triiodide complex, they are less irritating to the skin and have a shorter duration of antimicrobial activity than molecular iodine formulations like tincture of iodine or Lugol's solution. [26][27][28] Thus, the use of iodine as a pathogennonspecific intervention to prevent viral or bacterial infection typically entails a trade-off between the duration of its antimicrobial activity after application and the degree to which it irritates the skin to which it is applied.
3M™ Skin and Nasal Antiseptic (PVP-I solution 5% w/w [0.5% available iodine] USP) Patient Preoperative Skin Preparation Non-Sterile Solution (herein after abbreviated as "Nasal Prep") is a safe and effective formulation of 5% PVP-I with a proprietary filmforming composition that extends the duration of the iodine's antimicrobial activity. It has broad-spectrum microbiocidal activity against respiratory pathogens such as Streptococcus pneumoniae, 29 Haemophilus influenzae, 29 methicillin-susceptible and -resistant Staphylococcus aureus, 29 influenza A virus, 30 and coronaviruses, 30 including SARS-CoV-2. 31,32 Clinical studies 33,34 have shown that same-day application of Nasal Prep to the nares of preoperative patients is equivalent to or better than a 5-day course of intranasal mupirocin at preventing postoperative surgical site infections due to S. aureus. In addition, polymer and excipients in the Nasal Prep formulation protect PVP-I from inactivation by nasal mucins and other organic compounds and increase its adhesion to mucosal surfaces, 35 potentially imparting a longerlasting antimicrobial effect in the nose than other PVP-I preparations lacking these properties. We hypothesized that the PVP-I in Nasal Prep, applied intranasally, would prevent influenza virus infection in a guinea pig model of influenza virus transmission, as had been previously shown in mice that had tincture of iodine applied to their snouts. 25 We hypothesized further that the film-forming property of Nasal Prep would maintain its antiviral activity over a longer duration than with standard aqueous PVP-I solutions (typically 30-60 min), 27 thus enabling a practicable, once-daily reapplication interval.

| Animals
Female Hartley strain guinea pigs weighing 300-350 g were obtained from Charles River Laboratories. Animals were allowed free access to food and water and kept on a 12-h light/dark cycle. Guinea pigs were anesthetized prior to intranasal inoculation and to nasal washing, using a mixture of ketamine (30 mg/kg) and xylazine (5 mg/kg), admin- transmission experiments, strict measures were followed to prevent any cross-contamination between animals, including handling recipient animals before donors and changing gloves between guinea pigs.

| Application of nasal treatments
Nasal Prep (3M™ Skin and Nasal Antiseptic), a commercially available 5% (w/w) PVP-I solution, was used in these studies. The vehicle comparator (Nasal Prep devoid of PVP-I and sodium iodide) was produced by 3M specifically for these experiments, and the phosphate-buffered saline (PBS) comparator was purchased (Gibco). All three treatments were applied to guinea pig nares in the same way: While guinea pigs were awake and in an upright position, we used a positivedisplacement pipette to deliver 50 μl to each naris (100 μl per animal).  that the wire grids opposed each other at a separation distance of 5 cm; such an arrangement allows air to flow between cages but prevents contact between guinea pigs. Guinea pigs were kept together for a total of 7 days. Nasal washing was performed on Days 1, 3, 5, and 7 post inoculations by instilling a total of 1 ml of PBS + P/S into both nares and allowing it to drain onto a sterile Petri dish. Nasal treatments were reapplied every 24 h. Nasal wash samples were collected in 1.5-ml tubes on ice, centrifuged to pellet debris, and stored at À80 C until titration by plaque assay, as previously described. 44 45,48,49 (Text S1). Because the result of interest is the proportion of successful transmission events in each treatment group, the Bayes factor for independent multinomial sampling was calculated as a test for equality of proportions, with the row (treatment) margins fixed. 45 The magnitude of the Bayes factor quantifies the strength of evidence in support of the alternative hypothesis over the null hypothesis. For example, a Bayes factor of 5 means that, given the data observed, the alternative hypothesis is five times more likely to be correct than the null hypothesis, while a Bayes factor of 0.2 (the reciprocal of 5) indicates that the alternative hypothesis is five times less likely than the null hypothesis. 45 Bayes factors, being essentially odds ratios, can alternatively be expressed as the percent likelihood, from 0% to 100%, that a given hypothesis is likely to be the correct one, given the observed data (i.e., the posterior probability). 48

| RESULTS
To assess the effect of topical Nasal Prep applied prophylactically in the nares of guinea pigs intranasally inoculated with influenza virus, we compared Nasal Prep to two controls, the Nasal Prep "vehicle"  . This Bayes factor indicates that, given the observed data, the alternative hypothesis is only slightly favored over the null: There is a 57% likelihood that the transmission rates are truly different between Nasal Prep-and PBS-treated guinea pigs and, correspondingly, a 43% likelihood that they are not truly different; thus, these data provide insufficient evidence to meaningfully support either hypothesis over the other. 45 In the airborne model, Pan/99 is typically transmitted from untreated donors to 75% to 100% of untreated recipients 4,51 ; thus, while the 50% transmission rate in the PBS group is lower than would be expected if PBS treatment of both animals had no effect on transmission, the small group size provides insufficient evidence to conclusively reject the null hypothesis (Bayes factor = 2.84, which corresponds to a 26% likelihood that the transmission rate obtained in the PBS group is not actually different from the rate historically observed with this virus in untreated guinea pigs).  Figure 5). To reduce animal use, we did not include a PBS control group because it had been relatively ineffective at preventing transmission in the airborne model and thus would be expected to be equally as or even more ineffective in a contact model. 4,51 This experiment otherwise followed the same protocol as prior ones (Figures 3 and 4). Both Nasal Prep ( Figure 5A) and its iodine-free vehicle ( Figure 5B Figures 1 and 3 (Figure S1). That the curves happen to look similar to each other is, we speculate, due to random chance in a small number of animals. Prep was efficacious in reducing the frequency of virus transmission to recipient guinea pigs when the treatment was applied to virus recipients up to 24 h after they were exposed to infected donor guinea pigs in the same cage. These results, though preliminary, indicate that further investigation of Nasal Prep as a broad-spectrum intranasal antiseptic to prevent the transmission of influenza and other respiratory viruses is warranted.

However, Nasal Prep treatment of both donors and recipients
Interestingly, the vehicle alone, without PVP-I, also reduced influenza virus transmission in our model. The mechanism remains to be elucidated, but hypotheses include its pH being unfavorable for virus infectivity and/or its viscosity physically impeding the virus particle from reaching or interacting with host cells. Others have demonstrated that reducing the burden of Gram-positive bacterial nasal flora by preinoculation mupirocin decolonization impairs the transmissibility of influenza virus in ferrets, 52 which may also be contributing to the relative inefficiency of influenza virus transmission in Nasal Prepand perhaps also vehicle-treated guinea pigs. The number of transmission pairs in these experiments lacks sufficient statistical power to discern whether PVP-I, which has been shown to be virucidal for influenza viruses, 18 confers an additive benefit to the Nasal Prep vehicle alone in preventing influenza virus transmission in this model. It is, however, well known that enveloped viruses, such as influenza virus, are significantly easier to inactivate than non-enveloped viruses and many bacteria. 53  these factors must be studied in greater depth before this intervention could be deployed in humans. Despite these potential limitations, these experiments provide a strong rationale for further investigation of Nasal Prep as a pathogen-nonspecific intervention that may reduce the risk of respiratory virus infection including, potentially, future zoonotic viruses that have yet to manifest in humans.

CONFLICT OF INTEREST
Dr. Bouvier reports that she is an employee of the Icahn School of Medicine at Mount Sinai, which received funding from 3M Company during the conduct of the study. Dr. Landgrebe, Dr. Parthasarathy, and Dr. Wlashin report that they are employees of 3M, which received funding from DARPA during the conduct of the study. 3M employees conceived of the study and contributed to experimental design but had no direct participation in the conduct of the experiments or the collection or analysis of the data. Dr. Landgrebe, Dr.
Parthasarathy, and Dr. Wlashin also report three patents pending that are relevant to this work, entitled "Treatment and Prophylaxis of Viral F I G U R E 7 Preinoculation treatment of only donor guinea pigs in the contact transmission model. (A) No transmission was observed in four untreated recipient guinea pigs exposed to infected, treated donor guinea pigs. (B) Four transmission events were observed in four recipients exposed to infected, treated donor guinea pigs. Dotted lines/open symbols represent nasal wash virus titers of donor animals; solid lines/closed symbols represent nasal wash virus titers of recipient animals. LOD, limit of detection Infections," "Transmission Prevention of Viruses with Application of Antiseptic Composition," and "Virus Transmission with Application of a Composition." Dr. Gaaloul  Kevin D. Landgrebe: Conceptualization; funding acquisition; resources.

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1111/irv.13035.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.